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WO1997045743A1 - Procede pour selectionner des substances inhibitrices pathogenes cibles et trousses d'analyses prevues pour etre utilisees avec ces substances - Google Patents

Procede pour selectionner des substances inhibitrices pathogenes cibles et trousses d'analyses prevues pour etre utilisees avec ces substances Download PDF

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Publication number
WO1997045743A1
WO1997045743A1 PCT/FI1997/000339 FI9700339W WO9745743A1 WO 1997045743 A1 WO1997045743 A1 WO 1997045743A1 FI 9700339 W FI9700339 W FI 9700339W WO 9745743 A1 WO9745743 A1 WO 9745743A1
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WIPO (PCT)
Prior art keywords
seq
virus
pathogen
neutralizing
peptide
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PCT/FI1997/000339
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English (en)
Inventor
Hilkka Lankinen
Tuomas Heiskanen
Antti Vaheri
Åke LUNDKVIST
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Helsinki University Licensing Ltd.
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Application filed by Helsinki University Licensing Ltd. filed Critical Helsinki University Licensing Ltd.
Priority to AU29650/97A priority Critical patent/AU2965097A/en
Publication of WO1997045743A1 publication Critical patent/WO1997045743A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses

Definitions

  • the present invention is related to a method for selecting pathogen inhibiting substances with high affinity and neutralizing effect on pathogens and to find analogues to neutralizing substance useful as substances active against and in diagnostics of pathogens, especially against viruses, including hantaviruses and respiratory syncytial virus (RSV) or as leader molecules useful in comparative drug design as well as in pathogen diagnostics.
  • the invention is especially related to Puumala virus inhibiting ligands as well as to test kits useful for carrying out the method.
  • Random peptide libraries displayed on a filamentous phage represent a convenient pool of mutations or ligands with a multitude of variables and can be used as a source for many biologically active peptide ligands.
  • the affinity and activity of the ligands to the target is easy to evaluate by known phage display techniques and methods in cell, virus, bacteria and phage propagation.
  • the phage display technique based on affinity-purification of phage clones on target molecules has been applied for the selection of ligands or peptides with a high affinity to the binding sites of proteins or other macromolecules.
  • Such phage display techniques have been used for mapping epitopes of monoclonal antibodies, for identification of biologically active peptide ligands to different molecules and for determination of active domains of proteins (Scott and Smith, 1 990; Yayon et al., 1 993, Koivunen et al., 1993; Pasqualini et al., 1 995).
  • In the International Patent Publication WO 91 /1 8980 methods and compositions for identifying biologically active molecules are described.
  • Meulemans et al., 1 994 described phage-displayed antibodies obtained by using fixed cells and monoclonal antibodies as the eluting agent.
  • the International Patent Publication WO 96/04557 discloses a method for screening a phage displayed antibody library against target molecules, such as receptors, channels, transport and adhesion proteins to find small molecule pharmacophores.
  • affinity based screening is used.
  • Hong and Boulanger ( 1 995) demonstrated that functions of adenovirus, a non-enveloped virus, can be investigated with a phage-displayed peptide library using affinity elution combined with ligand elution.
  • the so called natural ligands in said publication are recombinantiy produced structural parts of the adenovirus.
  • Hong and Boulanger ( 1 995) show that known adenoviral structures can be used to replace attached phage-particles.
  • leader molecules which not only have a great affinity, but also mimic the functions of known neutralizing substances, i.e. they can prevent infection by analogous mechanisms.
  • no previous knowledge of the pathogen receptor and its structure is needed and it is possible to characterize several binding pockets on the pathogen without any previous knowledge of its structure.
  • Phage display techniques have demonstrated that biopanning selects against primary structures of the library. However, selection does not necessarily provide any analogues to naturally occurring pathogen inhibiting sequences. Only peptides with great affinity to the pathogen are obtained. In order to obtain a drug against a pathogen a desired specificity or target function is required. Pathogens can be neutralized e.g. by ligands directed to the specific cell receptor binding sites or other functional domains on the pathogen, and by ligands, which bind the pathogen and thereby indirectly inhibit its essential function(s).
  • binding pocket is on a highly variable region of the pathogen, it is highly probable that mutations, which create resistance, can occur in the pathogen. In such cases, it can be assumed that drug resistance is developed very rapidly against the chosen ligand and the pathogen is capable of escaping the effect of the new drug. In such cases there would be a continuous need to develop new variants of drugs.
  • leader molecules are selected among such compounds, which are directed against a site, which is not prone to variations and might be used in comparative studies against a broad spectrum of related organisms.
  • the method of the present invention allows easy screening of compounds for testing of escaping mutants.
  • the present invention also allows the development of combinatorial ligand libraries for further selections.
  • the conserved motifs obtained as a response to the selective pressure with the neutralizing substance might be shared by related pathogens, i.e. members of the same genus or family as the target pathogen or other pathogens, which carry same functional domains.
  • the present invention provides a method of screening target pathogen inhibiting ligand structures common for the whole genus or family of the target pathogen or ligand combinations with the same activities.
  • the present invention by using not only an affinity selection, but also a secondary selection with pathogen neutralizing substance(s) mimicks the selection process in nature to obtain a pool of functional analogues (compare polyclonal antibodies).
  • secondary structure analogues and primary structure motifs are obtained. These structures can be compared with structures and motifs obtained from the selections against other members of the pathogen genus or family.
  • a conserved structural motif obtained as a response to the selective pressure that was applied by the neutralizing substance might be shared by related pathogens or other pathogens that carry the same functional domains.
  • the present method provides a method for screening and selecting target pathogen inhibiting ligand analogue structures, which may have a broad activity.
  • the objective of the present invention is to provide an easy method for selecting large amounts of prescreened ligands on target pathogens and essentially intact parts of said native pathogens, in order to find such ligands, which are useful in comparative studies to find leader molecules for drug development against pathogens, related or non-related.
  • the method of the present invention comprises a competitive screening method, wherein native target pathogens, especially viruses such as Puumala or RS-virus or essentially intact parts thereof are attached to a solid carrier for example on a microtiter plate.
  • a ligand library is allowed to react with the bound target pathogen and the pathogen bound ligands are subjected to competitive affinity-elution with at least one neutralizing substance.
  • This step can be repeat ⁇ ed by one or more similar cycles of affinity enrichments with the eluates from the previous cycles until no improved affinity is found.
  • the pathogen inhibiting capability of the collected molecules are compared.
  • the present invention is related to compounds and pharmaceutically active compositions comprising parts or repeats of the following sequences HWMFSPW (SEQ ID NO: 1 :), CHWMFSPWC (SEQ ID NO:2:), PWYFQPW (SEQ ID NO:3:), CPWY- FQPWC (SEQ ID NO:4:) PWYFHPY (SEQ ID NO: 1 7:) and CPWYFHPYC (SEQ ID NO: 1 8:) .
  • the sequences are potentially useful as inhibitors of Puumala virus or as leader molecules for developing substances active against pathogens, especially enveloped viruses, such as hantaviruses and RS-virus inhibitors.
  • the present invention is also related to compounds or pharmaceutical compositions containing parts or repeats of the following sequences: LFNWPVN (SEQ ID NO:5:), CLFNWPVNC (SEQ ID NO:6:), SWIFWY (SEQ ID NO:7:), CSWIFWYC (SEQ ID NO:8:) , WIFWVY (SEQ ID NO:9:), CWIFWVYC (SEQ ID NO: 1 0:), HWSFGIF (SEQ ID NO: 1 1 :), CHWSFGIFC (SEQ ID NO: 12:), PWYLPW (SEQ ID NO: 1 3:) , CPWYLPWC (SEQ ID N0: 14:), PWYFFDL (SEQ ID NO: 1 5:), CPWYFFDLC (SEQ ID NO: 1 6:), PWYFHPY (SEQ ID NO: 1 7:) , CPWYFHPYC (SEQ ID NO: 1 8:), SWIFWEY (SEQ ID NO:
  • the substances selected by the method of the present invention include at least one of the amino acid sequences, LFXXXXX (SEQ ID NO :36:), LFXWPXX (SEQ ID NO :37:), XXXXXPW (SEQ ID NO :38:), XWXFXXX (SEQ ID NO :39:), XWXFXPW (SEQ ID NO :40:), LFNWPV (SEQ ID NO :41 :), XXXWPXX (SEQ ID NO:42:) , XXXXGIF (SEQ ID NO:43:>, PXWXXXX (SEQ ID NO: 44:), PWXF (SEQ ID NO:53:), XWXF (SEQ ID NO:55:), HWXF (SEQ ID NO:56:), XWXFXPX (SEQ ID NO:57:), and/or parts or repeats thereof useful as leader molecules for developing substances active against enveloped viruses
  • the invention is also related to test kits for selecting substances according to method of the present invention comprising a combinatorial ligand library consisting of at least one set of ligands obtainable from any of the cycles of the method of claim 1 , or parts or repeats of the substances or ligands, especially the substances mentioned above optionally combined with the original library in packaged combination with instruction for use.
  • a combinatorial ligand library consisting of at least one set of ligands obtainable from any of the cycles of the method of claim 1 , or parts or repeats of the substances or ligands, especially the substances mentioned above optionally combined with the original library in packaged combination with instruction for use.
  • Figure 1 shows a summary scheme of the ligand elution steps used in biopanning of Puumala virus with neutralizing monoclonal antibodies MAbs 5A2, 4G2 and 1 C9. Virus was attached on a well in Elisa plate and 3 consequetive affinity enrichments performed (pannings 1 -3). The MAb 4G2 enriched clones were finally purified in two acid elution steps (pannings 4-5) .
  • Figure 2A shows affinity and specificity of the indicated peptide inserts carrying phage clones for different domains of the envelope glycoproteins of purified Puumala virus (20 ng) as determined by competitive Elisa.
  • the competing monoclonal antibodies shown were added ( 1 ⁇ g/well) prior to the phage clones to all the other wells except those in column series one and wells coated with the blocking agent.
  • the competing MAbs included the neutralizing Puumala virus specific MAbs 1 C9, 4G2 and 5A2 and the non-neutralizing Puumala specific control MAbs G1 -e7-e5 and 1 C1 2.
  • the Elisa background was measured in wells that did not contain virus (BSA control) . Phages were select ⁇ ed by bank vole neutralizing MAb 4G2.
  • Figure 2B shows affinity and specificity of the indicated peptide inserts carrying phage clones for different domains of the envelope glycoproteins of purified Puumala virus (20 ng) as determined by competitive Elisa.
  • the competing monoclonal antibodies shown were added ( 1 ⁇ g/well) prior to the phage clones to all the other wells except those in column series one and wells coated with the blocking agent.
  • the competing MAbs included the neutralizing Puumala virus specific MAbs 1 C9, 4G2 and 5A2 and the non-neutralizing Puumala specific control MAbs G 1 -e7-e5 and 1 C1 2.
  • the Elisa background was measured in wells that did not contain virus (BSA control). Phages selected by human neutralizing MAb 1 C9 elution.
  • Figure 3A shows inhibition of binding of sequence LFNWPVN (SEQ ID NO:5:) carrying phage clone to Puumala-virus by synthetic cyclic peptides CLFNWPVNC (SEQ ID NO:6:) and CHEMFSPWC (SEQ ID NO:2:) .
  • the peptide CVRLNSLANC (SEQ ID NO:35:) was used as the negative control.
  • the background of 0.1 absorbance units has not been withdrawn from the values.
  • Figure 3B shows inhibition of binding of sequence HEMFSPW (SEQ ID NO:7:) carrying phage clone to Puumala-virus by synthetic cyclic peptides CLFNWPVNC (SEQ ID N0:6:) and CHEMFSPWC (SEQ ID NO:8:).
  • the peptide CVRLNSLANC (SEQ ID NO:35:) was used as the negative control.
  • the background of 0. 1 absorbance units has not been withdrawn from the values.
  • Figure 4 shows the inhibition of Puumala virus infection in Vero E6 cells by the synthetic cyclic CHWMFSPWC-NH2 (SEQ ID N0:2:) peptide.
  • pathogen means any infectious disease causing native organism or an essentially intact part of the native organism.
  • a "pathogen” can be a virus, bacterium, fungus or functionally intact parts of them.
  • the parts of the pathogens are preferably derived from native organisms.
  • the pathogen is an enveloped virus, from the family Bunyaviridae and Paramyxoviridae, but other viruses, including rotavirus, herpes virus, hepatitis virus, rhinovirus, enterovirus, and Hl-virus, but also non-enveloped viruses, such as adenovirus as well as functionally active parts thereof.
  • the pathogens include both enveloped, such as ortomyxo-, hepatitis B- and D-, Flavi-, Arena- and lentivirus, etc. and non-envelope viruses, such as rota-, adeno-, pikorna-, parvo-, hepatitis A-, entero-, coxsackie virus, rinovirus, etc.
  • the model target virus in the present invention is Puumala virus, a member of the hantavirus group belonging to the Bunyaviridae family.
  • Puumala virus which is a representative member of enveloped viruses, is used as a model target pathogen in the present invention for screening leader molecules, which are active not only to Puumala virus, but possible also to other related viruses, such as hantaviruses as well as other members of the Bunyaviridae-family.
  • Another promising model system has been developed with RS -virus. This shows that the method can be ubiquitously applied.
  • neutralizing substance means a compound, ligand or molecule with a special function, e.g. a neutralizing antibody function, which when it is bound to the pathogen or virus prevents infection.
  • the antibody for example, binds on a site of the pathogen, e.g. the virus envelope, which is essential for the attachment and/or penetration of the pathogen virus.
  • Said substance can be a receptor molecule, but it is preferably a polyclonal or monoclonal antibody, preferably raised against a native pathogen or parts of a native pathogen. In the present invention it is essential to use neutralizing antibodies, preferably raised against native pathogens. No other restrictions are connected to the antibodies. They can be raised with any conventional technique, including hybridoma techniques.
  • the "mechanism of neutralization” is generally provided by B-cell immunity and not by cell mediated immunity.
  • the mechanism can include the binding of an antibody to a site on the pathogen or virus that sterically hinders an essential function of the pathogen, in case the pathogen is a virus, e.g. virus attachment or penetration.
  • the antibody can bind to several sites on the virus and thereby prevent, in case the pathogen is a virus, protein components from functioning or maturing normally, e.g. in attachment, membrane fusion or penetration.
  • Another “mechanism of neutralization” is the binding of an antibody to a site on the pathogen separate from a functional domain, which causes a structural change in the pathogen. This leads to inhibition of the normal functions e.g. attachment or fusion of a virus.
  • the mechanism can also be the binding of an antibody to a site on the pathogen or virus that is unrelated to the functions of attachment, fusion or penetration, but instead inhibits the pathogen or virus after its internalization by intervening with some later essential function, e.g. virus uncoating.
  • a “mechanism of at least partial neutralization” can be described as the binding of an antibody on the pathogen or the virus resulting in its recognition by immune defense, e.g. macrophages, which internalize the pathogen or virus trough immunoglubulin Fc-domain and degrade the pathogen or virus or complement, which by recognition of immunoglobulin destroys the cell infected by the pathogen (clearance).
  • neutralizing antibody can be either monoclonal or polyclonal.
  • a “neutralizing monoclonal antibody” comprises a homogeneous immunoglobulin molecule with binding properties normally restrictive to one single epitope or binding site on the antigen.
  • a “neutralizing polyclonal antibody” is a heterogeneous mixture of immunoglobulins or their so called Fab fragments, the part of the Ig that interacts with an antigen divalently, i.e. has two identical arms for binding the epitope, and can be produced from Ig by proteolytic cleavage.
  • Fab-fragments of antibodies can also be produced by cloning.
  • a phage library application can be developed for the production. Said Fab-fragments all have the same specificity defined as inhibition of infection or prevention of infection.
  • Neutralizing polyclonal antibodies can bind to one or several epitopes or binding sites.
  • neutralization site on a pathogen or virus means a locus or domain, on which an antibody can bind and by doing so prevents infection. Structures of "neutralization sites” resemble antibody epitopes and can be found in four different categories, which are described below.
  • a “continuous epitope” means an epitope composed of amino acid residues, which are proximal in the primary structure.
  • An antibody fits a peptide or parts of a protein, which is about 1 3 amino acid long (structural epitope), whereas a binding can be demonstrated with a minimum of 3 residues (functional epitope) .
  • a “conformational epitope” or a “discontinuous epitope” is composed of different parts of a protein and is dependent on its secondary (x-helixes, O-sheets, turns) and tertiary structures (protein domains composed of x-helixes, P-sheets, turns) .
  • a "cryptotope” is a hidden epitope, which becomes exposed after the protein has become denatured, fragmented, depolymerized, etc.
  • Virus capsids often contain "cryotopes" and therefore an intact pathogen or viral particle is a benefit in initial screening, since production of antibodies during infection or otherwise may lead to exposure of such potential epitopes, which in a situation of a pathogen, especially a virus challenge might be useless or even harmful targets.
  • a “neotope” is an epitope that is found only in quaternary structures of proteins, but not in protein monomers. The conformational structure of a monomer changes in a quaternary complex and this leads to creation of an "epitope” . "Neotopes” are again found in viral capsids.
  • combinatorial therapies means that the pathogen inhibiting substance(s) are active based on at least two functions or principles.
  • prophylactic principle relies on “virus neutralization” on first hand and de ⁇ velopment of synthetic vaccine in the second hand.
  • wash means incubating, including washing or rinsing of the solid carrier or adsorbent for removal of its adsorbate.
  • competitive elution or “equilibrium elution” means incubation with a substance or ligand, e.g. neutralizing substance, which competes out the ligands attached to the solid carrier by affinity and concentration for the solid phase bound target binding site.
  • a substance or ligand e.g. neutralizing substance
  • ligand elution means incubation and/or rinsing or washing of the adsorbent for removal of the adsorbate, specifically by a competition mechanism for the binding site interaction, in which the eluent or neutralizing substance replaces the eluate or bound members of the ligand library.
  • replacement is a concentration dependent affinity (binding strength) of the members of the ligand library or eluent, i.e. the neutralizing substance for the binding site. "Replacement” is determined by the affinity of the eluent or neutralizing substance for the binding site versus avidity of the eluate or bound members of the ligand library for the binding site. The efficiency of "replacement” is highly dependent of and determined by the concentration of the eluent or neutralizing substance. In other words "replacement" of ligand bound to the pathogen is more efficient, when the concentration of the neutralizing substance is higher and/or the affinity of the neutralizing substance is stronger, than the concentration and affinity of the bound ligand.
  • non-specific physicochemical elution means an incubation with substance with low or high pH or with polar solvents, including blocking agents, mock antibodies, etc.
  • ligand means a natural counterpart of a neutralizing substance, antibody or receptor of the target pathogen, for which the target pathogen has a specific binding site.
  • ligand library means a library of polymer- or oligomer-forming substances or specificities of corresponding origin.
  • libraries are for example, amino acid analogues, e.g. their D-forms, sugar molecule libraries, but in the present invention it is preferably a peptide library or a glycopeptide library, optionally displayed on a phage or an eucaryotic vector. No particular library is required in the present invention. On the contrary, it is an advantage if different types of libraries of different origin can be used. It gives an improved opportunity of finding different pathogen inhibiting substances.
  • the sugar library can be prepared from oligosaccharides comprising different sets of sugar monomers or be presented as glycopeptide libraries produced e.g. in eucaryotic expression systems.
  • the "ligand library” is most preferably a peptide library, which comprises synthetic, semisynthetic or natural peptides. Recombinant DNA techniques can also be used to provide the peptides.
  • the amino acid sequences used in the peptide library have the formula -(X)n-. Optionally, they are flanked by cysteine residues.
  • X means any amino acid and n is an integer of 3-40, preferably 4-20, most preferably 5-10.
  • the smallest useful peptide comprises 3 amino acid residues. Peptides containing less than 3 amino acid residues are not capable of fully neutralizing the pathogen target. The maximum size is about 20-40 amino acids. Such peptides may be too large to fit into or onto some functionally active essential intact domains of the target pathogen.
  • Peptide libraries containing 7-1 2 amino acids might be optimal and preferably the peptide motif should consist of anything between 4 and 12 amino acids, most preferably 6, 7 or 8 amino acids.
  • the "combinatorial ligand library” especially means a library comprising of at least one set of ligands obtainable by the method of the present invention or other prescreened libraries or reconstructed libraries and/or any combinations thereof, optionally combined with the original library.
  • these "combinatorial ligand libraries” can be combined with molecules that have separate functions or as components of chimeric molecules.
  • the “solid carrier” means a microtiter plate or an affinity column.
  • Other solid and/or porous supports, such as beads, biosensors, sticks and plates with tags or pigs can be used as well.
  • the BIA-technology Biological Interaction Assay developed by Pharmacia is one possible application.
  • the matrix of this biosensor is called sensor chip.
  • the present invention is developed based on an application of per se known biopanning and phage display techniques.
  • the invention is related to a method for screening pathogen inhibiting, especially virus inhibiting ligands by application of competitive selection pressure.
  • the method is potentially useful for finding target pathogen inhibiting substances with a high affinity and neutralizing effect.
  • Said substances are useful as potential pathogen inhibiting drugs or for development of therapeutically active, protective, prophylactic and/or diagnostic substances as well as combinatorial therapies against pathogens, such as viruses (including strains, groups and/or families).
  • the invention is a method for identifying composite functional and structural mirror images of neutralizable domains on pathogens, from which by using stepwise competitive selection pressures presence of desired functions can be tested out.
  • the corresponding structures and their freedoms are found by functional and structural characterisation of said image forming substances.
  • the invention describes ligands active as antagonists, agonists, stimulators or possibly as other effectors in enveloped viruses, such as RS-virus and hantavirus, especially Puumala virus infections.
  • the invention also describes substances and compositions, especially peptides, which bind to Puumala virus neutralization site and further structural motifs potentially representing evolutionary constraints and principles to bind other hantaviruses and possibly all Bunyaviridae viruses as well as general known and yet unidentified functions, principles and mechanisms for selection of peptides by competitive elution.
  • target pathogens and ligand libraries from which ligands with antipathogenic activity can be selected are the first steps in development of a drug function.
  • Suitable target sites or domains for example on a virus, are found from essential steps of virus replication
  • Useful target pathogen functions specific for viruses can be found by analysing the essential steps of virus replication including attachment, penetration, uncoating, gene expression, assembly, maturation and release of the virus.
  • Most of the presently used antiviral target functions involve nucleic acid synthesis or other intracellular steps of the virus replication cycle, whereas virus entry, which may offer benefits over the other target functions is less exploited probably because of drug resistance.
  • Suitable assay systems have been created based on the present invention, pharmaceutical companies can facilitate identification of potential antiviral compounds for further evaluation, e.g. by random screening of a multitude of pathogens, ligand libraries and neutralizing substances.
  • a promising application in the random search of antivirals are the so called combinatorial peptide libraries; synthetic or recombinant DNA peptide libraries that are employed in identification of bioactive peptides.
  • Attachment of pathogen can be blocked both by ligands directed to the cell re ⁇ ceptor of said pathogen and by such, that only bind to the pathogen.
  • Our rational to select an essentially intact native target pathogen particle was that we may thereby find mimotypic ligands, which do not intervene with cellular functions.
  • Random ligand libraries displayed on a filamentous phage can represent such a pool of mutations and has been used as a source for e.g. many biologically active peptide ligands.
  • the phage display method is based on affinity-purification of phage clones on a target pathogen and should select from the ligand library those ligands, which attach with the highest affinity to the binding sites of the pathogen.
  • the most effective and straightforward way to enhance and specify the affinity purification of a ligand library component is to competitively elute the relevant members of the ligand library attached to the solid carrier with a neutralizing substance, which has the desired biological activities.
  • the selected ligands or molecules provide a tool to study the interaction between target and neutralizing substance and may even facilitate molecular modelling of other inhibitors, i.e. target pathogen inhibitors provide leader molecules for designing drugs.
  • Affinity elution provides shortcuts to identification of neutralizing sites on the target pathogens and gives the structure of the active core structure of the neutralizing substance.
  • the yield of primary sequences by competitive selection pressure should include all the interaction freedoms that are shared between the target molecule and the ligands, such as peptides present in the library and capable of binding to the neutralizing site of the target pathogen.
  • the present invention provides a method for selecting pathogen inhibiting ligands potentially useful as leader molecules in drug development by screening ligand libraries against a selected target pathogen by competitive elution with a neutralizing substance.
  • the method provides a powerful tool for developing therapeutically active, protective and prophylactic or diagnostic substances.
  • a pathogen or a functionally active essentially intact part of a target pathogen is chosen.
  • the pathogen is selected from a group or family of a virus, bacterium or fungus, including intact functional parts thereof.
  • the method is particularly applicable for selecting virus inhibiting ligands against enveloped viruses.
  • the present invention provides two principal selection pressures against the library.
  • a primary selection which is against specific binding characteristics of the ligands or peptides and the secondary selection against residues that are important in the interaction of the neutralizing substance and target pathogen. Since both the target pathogen binding sites and the target pathogen ligand interactions occur on constrained surfaces, which are determined by secondary, tertiary and quaternary structures, the primary selection occurs against structural constraints of peptides.
  • the theoretical number of freedoms in the present screening method is the same as the number of peptide binding sites or pockets on the pathogen surface and the aim of the method including the competitive elution is to specifically define those pockets or sites.
  • a well chosen library should cover all the binding freedoms that will dissect the primary structure elements of the target pathogen ligand interaction. It is not certain that the freedoms of primary selection include any composition which would lead to the desired biological activity since biological activities of proteins tend to involve also other than structurally important residues.
  • the secondary selection is against residues that are important in the interaction of the target pathogen and its neutralizing substance, but not or not alone for binding of the peptide.
  • the ligand library is exposed to a system that imitates the selective pressure in nature and as a result a set of ligands are obtained, which act on the functionally active part of the pathogen target.
  • they provide an image of the active site of the target pathogen.
  • This allows the neutralizing site of the target pathogen to be identified and characterized functionally.
  • the collected ligand structure function information can be used to design drugs for related members of the target pathogen group or family. It is naturally possible that only such residues, which are critical for neutralization of the target pathogen are obtained in the secondary selection.
  • the present invention is also related to a method for characterizing the mimo ⁇ tope of the neutralizing site comprising one or more cycles of attachment-elu- tion-amplifications.
  • the target pathogen or a functionally active, essentially intact part thereof is attached to a solid carrier and is allowed to react with a molecule library.
  • the pathogen binding molecule is eluted by affinity-elution with at least one neutralizing ligand until the affinity enrichment is completed. Thereafter, the pathogen binding molecules of the last cycle are collected. Their structure and inhibiting effect is determined and their consensus sequences are checked. The information collected on the functional and structural data is combined to obtain the mimotope structure.
  • the present invention it is possibly to characterize the functionally active domain (site) on the target pathogen.
  • the site has a high affinity to a peptide of the formula described above and can be defined as a mirror image of the com ⁇ pound selected by the screening method for the present invention.
  • the method imitates the selection pressure in nature and allows the user to identify the target and the target binding substance both functionally and structurally.
  • a preselection is performed by allowing the said selected target virus to react with a peptide library. After washing and eluting the solid carrier bound amino acid sequences with at least one first mock-washing antibody and at least one second neutralizing antibody and the second elute is collected;
  • the second elute is optionally amplified and the titers thereof is determined;
  • the high affinity virus-binding peptides are selected by performing at least one preferably at least two affinity enrichments by affinity-elution described in step (b) with the elutes, which gave the lowest concentration of virus;
  • the high affinity virus-binding peptides are collected by performing one or more optional non-specific elutions. Any remaining antibody is optionally destroyed with a suitable washing substance, e.g. Prosep A followed by a repetition of the non-specific elution without any washing step; with antibody destroying agents; and as the last step
  • the structure is determined by sequencing the specifically eluted clones for identification of their target binding characteristics and inhibiting capability to select those ligands, which compete with the neutralizing substances and are both structural and preferably also functional analogues of said neutralizing substances.
  • Said ligands are potentially useful as pathogen inhibitors, as leader molecules or models for designing other pathogen inhibiting substances and to provide selected ligand libraries for screening other members of the groups, genera and families, related to the selected target pathogen.
  • Selected target pathogens or functionally active parts thereof are attached to a solid carrier in one or more concentrations.
  • the solid carrier with the target pathogen and a control without said target pathogen are blocked using bovine serum albumin, fat dry milk, Tween or other blocking agents, which do not intervene with affinity selection.
  • the ligand library presenting the peptides or other molecules is contacted with the solid carrier bound pathogen and said control and an interactions is allowed to take place. Non-bound ligands are washed away with a suitable buffer, which retains the structural integrity of the target pathogen without any preference.
  • the buffer used has a neutral pH and contains physiological concentrations of salts and optional additives like other salts, detergents, blocking agents or other components, which facilitate removal of unspecifically bound ligands, which have specific interactions with said target. Washing away undesired, bound molecules with a selective eluent is optional. An affinity elution, is performed, in which said target pathogen bound, washed ligands are competed out with a neutralizing ligand to be used in the screening method.
  • the structure analysis of the eluted ligands must be performed. If a phage ligand library is used phage titration of the phage elutes and the control from 1st panning is performed. The elute having the lowest target pathogen concentration and which has a phage titer higher than the control is selected and with the selected elute(s) an amplification of the phage clones of the elute is performed and an optional extra purification to remove any undesired ligand contamination is made.
  • Said steps are repeated at least twice using the amplified elutes.
  • Phage titers of said eluates and controls are compared in order to obtain an enrichment factor after the 2nd panning.
  • the phage clones of the eluates having the lowest target pathogen concentration and a phage titer higher than the control are selected for repeated selection processes and the enrichment factor compared with the 1 st and 2nd panning is evaluated.
  • the competitive affinity elution is repeated as long as said enrichment factor rises.
  • the number of cycles and combinations with other elutions than said elution with the neutralizing substance is optional.
  • Unspecific elutions should be used to assist the elution with the neutralizing substance in order to select clones, which have neutralizing substance specifici ⁇ ty and high affinity. Many alternative possibilities are available.
  • Competitive elution with neutralizing substances can be combined with non-specific elution, such as acid elutions for selection of clones that bind more tightly than those obtained by neutralizing substance elution alone. Acid elution can be performed in parallel with neutralizing substance elution and results can be compared.
  • Non-specific physicochemical elution such as an acid elution, can be performed after each neutralizing substance elution of the remaining clones.
  • Alternating neutralizing substance elutions and acid elutions can be made.
  • the ligands are collected with one or more non-specific elutions.
  • One or two non-specific elutions can again be followed with one or more competitive neutralizing substance elutions.
  • the present invention also provides a system or facilities for screening the pathogen inhibiting ligands, which can be used for identifying therapeutically active, protective and prophylactic substances.
  • the system comprises the preselected, purified target pathogen or a functionally active essentially intact part thereof attached to a solid carrier or support. It also comprises a ligand library, preferably displayed on a filamentous phage.
  • a ligand library preferably displayed on a filamentous phage.
  • One or more selective pathogen neutralizing substances and means for performing the determination needed are also provided.
  • a system for determining the concentration of the pathogen, phage, ligand, etc. as well as means for propagation of cells, bacteria, viruses and phages.
  • a system for screening the inhibitory effect of the high affinity ligand on the target pathogen is also provided.
  • the invention is also related to test kits for selecting substances according to method of the present invention comprising a combinatorial ligand library consisting of at least one, set of ligands obtainable from any of the cycles obtainable by the method of the present invention and/or parts or repeats of said substances or ligands, optionally combined with the original library in packaged combination with instruction for use.
  • the test kit can optionally contain target pathogens with or without solid-carriers, neutralizing substances, solvents, diluents, etc.
  • the Puumala virus was used as a virus model, but the principles disclosed are applicable to any group of virus, especially enveloped viruses.
  • the phage clones inhibitory for Puumala virus infection were enriched by competitive elution with virus neutralizing monoclonal antibodies.
  • the method applies to any pathogen as development of neutralizing antibodies is a general defense mechanism of nature against the spreading of infections.
  • Puumala virus belongs to the genus Hantavirus, family Bunyaviridae.
  • Hanta vi ⁇ ruses are enveloped and have a trisegmented, single-stranded, negative sense RNA-genome, the large (L), medium (M) and small (S) segments are packed to nucleocapsids separately.
  • L-segment encodes the viral RNA polymerase/tran- scriptase, M the two envelope glycoproteins Gl and G2, and S the nucleocapsid protein N (Elliott, 1 990, Kolakofsky, 1 993) .
  • Each hantavirus is associated with a primary rodent host carrier in which it has evolved (Plyusnin et.al., 1 994) .
  • Hanta viruses include human pathogens of two diseases with different clinical symptoms; hemorrhagic fever with renal syndrome (HFRS) is caused by Hantaan, Seoul and Puumala viruses (Schmaljohn et al, 1 985; Lee et al. , 1 990) and the highly lethal hantaviral pulmonary syndrome (HPS) by Sin Nombre and related virus strains (Nichol et al, 1 993; Zaki et al, 1 995; Hjelle et al, 1 994) .
  • HFRS hemorrhagic fever with renal syndrome
  • HPS highly lethal hantaviral pulmonary syndrome
  • Puumala virus is the etiologic agent of nephropathia epidemics, which is a mild form of HFRS and the carrier of Puumala is bank vole or Clethrionomys glareolus of Arvicolinae family (Schmaljohn et al., 1 985).
  • Phylogenetically Puumala virus is more related to HPS-causing viruses, also of Arvicolinae rodents, than are the other HFRS-causing viruses, Hantaan and Seoul of Murinae rodents. (Spiropolou et al, 1 994) .
  • the markers of hantaviral genome that result in pathogenicity and its divergences are unknown.
  • the pathogenicity of HFRS includes e.g. epithelial lesions and a variety of symptoms that propose involvement of the immune system. The virus infects cells from many organs and replicates slowly (Temonen et al., 1 993) but the virus-cell interactions are poorly understood.
  • the present projects stems from the original discovery in 1 980 of a major pathogenic virus, Puumala virus, the causative agent of nephropathia epidemics from the bank vole (Clethrionomys glareolus) .
  • the peptide studies for the development of a hantaviral drug was started in 1 991 as a program of molecular control of hantavirus infections.
  • vaccines against Hantaan and Seoul virus infections are being developed, large scale vaccination programmes may not be realized and especially not with all hantaviruses, because of the endemic nature of the diseases. Availability of a drug is desirable against these life threatening infections.
  • the antiviral agent ribavirin that has a broad antiviral activity has an effect also on hantavirus infections, but more research is required to assess the potential of drug therapy in hantaviral disease. It is generally believed, that viraemia is passed at the onset of HFRS, since virus and particularly Puumala virus is difficult to isolate from human urine. However, nephropathia epidemica patients, who die or get a severe shock secrete virus.
  • the panning reaction in a microtiter well is divided in a liquid and a stationary phase.
  • the amount of phage clones attaching to the stationary phase becomes lowered and thereby specificity of obtained phage clones increased.
  • the first case the binding of phages to certain binding sites on the target molecule is prevented.
  • the phages bind only to the sites of the target Puumala virus, which are differ ⁇ ent from the sites present in the related analogous molecule in the liquid phase.
  • a second way to increase the specificity of panning is to vary the washing and elution conditions. It seemed possible to elute selectively the phages, which attach to the same domains of virus as the neutralizing antibodies.
  • Affinity enrichment of the Puumala virus binding peptides with the MAb 4G2 and 1 C9 were successful in our biopanning scheme and lead to enrichment of three characterized classes of consensus sequences, which had four binding specificities and one motif XWFXPXC (SEQ ID NO: 57:) that is proposed to be essential for the peptide to inhibit Puumala virus infection.
  • Comparison of relative binding strengths of peptides propose a high binding consensus motif XXXXXPW (SEQ ID NO :38:) at the C-terminus and XWXFXXX (SEQ ID NO :39:) binding motif at the N-terminus.
  • the binding sites would thus need to be defined from the sequence XWXFXPW (SEQ ID NO :40:) , where positions 2, 4, 6 and 7 might determine both the binding and the inhibition specificity of the selection. Variation at positions 1 , 3 and 5 might lead to conformational changes that influence affinity. Positions 2 and 4 determine binding and position 6 inhibition.
  • the consensus sequences LFNWPV (SEQ ID NO :41 :) is interesting because of its complementary features.
  • the approach of functional mimotope mapping of Puumala virus neutralization epitopes could be extended as wide as the whole Bunyaviridae virus family.
  • the MAbs used in this study are specific to Puumala virus but it is expected that the same principles apply also to other hanta and maybe to the whole Bunyaviridae family.
  • the present invention provides a method to know whether a wide specificity inhibitor could be found and gives an added value to the approach.
  • the Puumala virus ligand selection provides a good test system, since the neutralizing antibodies are virus specific and the pathogen belongs to a virus family of long evolutionary history.
  • the primary sequence diversification is combined with conservation of structural elements in envelope proteins represented by conserved cysteines (Elliott, 1990) .
  • the selection provided may be specific to the particular library that was used. Although the most inhibitory sequence HWMFSPW (SEQ ID NO: 1 :) was found also from X2CX 1 2CX2 library.
  • One aim of the present invention is to provide new libraries for testing the sequences obtained on other viruses in other groups.
  • the ultimate goal of the screening method and the system of the present invention is to provide pathogen inhibiting ligands useful as antipathogenic drugs.
  • Leader molecules for developing other pathogen or virus inhibiting compounds either based on the structural information of the pathogen inhibiting ligand or experimental evidence of affinity to a neutralizing site on the pathogen.
  • the cyclic CHWMFSPWC-NH2 inhibited Puumala at 2.5 ⁇ M, the eluting antibody at 4 nM and the inhibitory sequence carrying phage clone at 1 nM. Suggesting that the phage acts as antibody and the peptide as one of its Fab-fragments.
  • Antibodies when they bind may move proteins, cross-link them, change their conformations and so on. The same can happen with the selected peptides that were made to compete just for said antibodies. They may perfectly mimic the antibodies or they may compete only for some properties of the binding critical residues.
  • Tables II and III show sequences and parts of them, which are useful in drug development, vaccine development, development of prophylactic strategies or diagnostic use.
  • the strategy of production of molecules that bind to the virus particle can be applied for any virus, especially enveloped viruses, and can be peptides selected, which that mimick interactions and functions of neutralizing antibodies or other ligands that interact with the virus in vivo.
  • the selected peptides may help to dissect from virus-cell interactions, those sites and residues, that are essential for virus attachment and entry and provide molecules, which specifically inhibit virus.
  • our results provide a proof for a methodology in approaches to antiviral agents.
  • the consensus sequence sets selected with Puumala virus for selection of Puumala, hanta or Bunyaviridae virus inhibitors are given in the text.
  • Antibodies, peptides, cells and virus Monoclonal bank vole and human Puuma ⁇ la virus antibodies used in this study were produced and purified with protein-G affinity chromatography as described (Lundkvist et al., 1 991 ; Lundkvist and Niklasson, 1 992; Lundkvist et al., 1 993a) .
  • Peptides were synthetized on a 433A Peptide Synthesizer (Applied Biosystems) using Fmoc -chemistry, cyclisized in dimethylsulfoxide (DMSO) as described (Domingo et al. , 1 991 ) and purified by reverse phase high performance liquid chromatography (HPLC).
  • Vero E6 cells were grown in Eagle's Minimal Essential Medium (MEM) supplemented with 1 0 % fetal calf serum (FCS), glutamine and antibiotics.
  • MEM Eagle's Minimal Essential Medium
  • FCS fetal calf serum
  • the Puumala Sotkamo strain virus was propagated in Vero E6 cells as described (Schmaljohn et al. , 1 985) and mass production of the virus was done in the same cells (Schmaljohn et al. , 1 983) .
  • the medium release of virus was followed up to 30 days post infection with hemagglutination tests and collected ea'ch second day on days 14 to 30 post infection, the collected medium was clarified by short centrifugation and stored on ice until all was concentrated using Pelican filter system (Millipore) .
  • the final concentration of the virus was done in a 20-60 % sucrose gradient in Tris HCl pH 7.6 buffer, the virus was inactivated with 0.05 % n-octyl ⁇ -D-glucopyranoside (Sigma) and the protein concentration of the virus preparation was determined in Pierce BCA protein assay to be in the range of 1 mg/ml.
  • the virus preparation was stored at -70 °C until use.
  • the filamentous phage cyclic heptapep- tide library inserted into the amino-terminus of the pill protein of the phage fd-tet was kindly provided by Prof. Erkki Ruoslahti (La Jolla Cancer Research Foundation, La Jolla, California, U.S.) and Dr Erkki Koivunen (Koivunen et al., 1 994) .
  • Affinity biopanning procedures were performed as described with some modifications (Smith and Scott, 1 993; Koivunen et al., 1 993) as schematically presented in Figure 1 .
  • a microliter well was coated with 1 .5 ⁇ g of mass purified, inactivated Puumala virus, strain Sotkamo diluted in Tris-buffered saline, pH 7.5 (TBS); 100 ⁇ l of virus was added per well, the plate incubated for one hour at 37 °C and blocked for one hour at 37 °C with TBS containing 1 % BSA.
  • TBST TBS containing 0.05 % Tween 20
  • 5x 1 O ⁇ transducing units (TU; 1 1 0 TU/clone) of CXyC-library was added to the virus-coated and the control well in 1 1 5 ⁇ l of TBS containing 3 % non-fat dry milk powder (NFDMP) and 5 mg/ml bovine serum albumin (BSA), and incubated for one hour at 37 °C.
  • NFDMP non-fat dry milk powder
  • BSA bovine serum albumin
  • Biopanning of Puumala virus with the cyclic heptapeptide library using MAb 1 C9 as the eluting antibody was done as described above for the 4G2 MAb with the following modifications.
  • the phages were allowed to bind Puumala virus in Dulbecco medium containing 0.33 % BSA.
  • envelope glycoprotein G2-specific MAb 5B7 was used for the mock elution prior to the 1 C9-elution (50 ⁇ g/ml) and the well was washed first with Dulbecco medium, 1 % BSA followed by a medium wash without BSA. Before the second panning any residual MAb 1 C9 was removed with Prosep A.
  • Binding assays The relative binding strength of phage clones and populations to the virus particle preparation was estimated in enzyme linked immuno-sorbent (ELISA) binding assay by peroxidase conjugated anti-MI3 antibody (Pharmacia) detection. All the incubations were performed in 1 00 ⁇ l volume and at 37 °C. Microtiter wells were coated for one hour with a suitable concentration of virus ( 10-20 ng/well) diluted in phosphate-buffered saline (PBS), blocked for one hour either with 1 % BSA or 5 % NFDMP in PBS, 0.05 % Tween 20.
  • ELISA enzyme linked immuno-sorbent
  • phages representing either one phage clone or a phage population was added to the wells in PBS, 0.05 % Tween 20 and the plate was incubated for 40-60 minutes.
  • the wells were washed five times with PBST and anti-MI3 horse radish peroxidase (HRP) conjugate (Pharmacia) in 1 : 5000 dilution of PBST was added, the plate was incubated for 30 minutes. After washing the wells eight times with PBST the remaining peroxidase activity was determined with 0-phenylenediamine dihydrochloride (0PD, Sigma) substrate at room temperature. The reaction was stopped after 10- 1 5 minutes with 100 ⁇ l of 2 M H2SO4 and the absorbance at 492 nm was measured.
  • HRP horse radish peroxidase
  • Antibody competition and peptide competition assays were performed using the ELISA-binding assay described above.
  • the competing peptides or antibodies ( 1 ⁇ g/well) were added to the virus coated wells 1 5 minutes prior to the phage clones or populations.
  • the phage samples in either PBS (50 ⁇ l) or Hank's ( 100 ⁇ l) were made up to 500 ⁇ l by addition of Hank's supplemented with 2-4 % FCS and 1 0-20 mM HEPES and overlayed on confluent Vero E6-cells on 6-well Limbro plates. The virus was allowed to absorb for one hour at 37 °C after which the wells were washed with PBS and the culture medium added.
  • the amount of expressed Puumala N-protein was measured in antigen capture ELISA essentially as described (Lundkvist et aL, 1 995) except that for the detection rabbit polyclonal anti-N-GST-fusion protein serum, diluted 1 : 1 000 (Vapalahti et al. , 1 995) was used.
  • the conjugate was anti-rabbit-immunoglubu- lin-HRP at 1 : 1 000 dilution, the peroxidase activity was monitored as described above.
  • a synthetic peptide was solubilized and serially diluted in Hank's balanced salt solution supplemented with 0.1 % BSA and 20 mM Hepes pH 7.4. Prior to infection 90 FFU of Puumala virus was added to the samples, which were incubated for one hour at 37 °C. Focus reduction neutralization test (FRNT) (Heiskanen et al., 1 997) was performed. The results are shown in Figure 4.
  • FRNT Focus reduction neutralization test
  • the antibody 5A2 and subsequently the MAb 4G2 were used for differential elution of phages bound on the same virus sample in a well.
  • the control well was coated only with BSA.
  • In the 5A2-eluate of the virus coated well there were approximately as many phages as in the 5A2-eluate of the control well, whereas in the 4G2-eluate of the virus coated well there was already an eight-fold enrichment of phages as compared to the control well.
  • the two antibodies are from the same species (bank vole) and belong to the same class, their constant domains are similar. Therefore, the detachment of phages by the 4G2-elution was likely to be due to the variable domain of the 4G2 MAb.
  • the sequences of the 4G2 selected clones may be divided into three main groups (Table II), in which there may be subgroups composed of either one or several different sequences, all depending on the level of homology. The frequency of each obtained sequence varied, the subgroups la and lb had only one representative each and the la sequence was the most frequent of all obtained. In both group lc and group Id the first four residues were conserved. In contrast, the group II sequences had hardly any resemblance to the group I sequences except that the consensus PWXF (SEQ ID NO:53:) of the group Id was reverted to F (N or T) WP in the group lla.
  • the single sequence in group lib was well represented in the clones selected with the MAb 1 C9 (Table III) where it showed a certain variation. All of the MAb 1 C9 selected clones were of the group lib. The group III sequence appeared once and diverts from all the other sequences found. In an overall comparison the most conserved residues are at positions 2 and 4 and the first four residues vary less than the following three. The sequences are in large hydrophobic pocket, rich in aromatic residues and proline, proposing a selection against a bulky fit into a hydrophobic pocket or pockets.
  • Relative affinities of the selected clones were compared in ELISA-binding assay titrations (data not shown) .
  • the phage clone with highest affinity for Puumala virus was in group lla with the sequence LFNWPVN (SEQ ID NO:5:) the inhibitory sequences HWMFSPW (SEQ ID NO: 1 :) and PWYFQPW (SEQ ID NO:3:).
  • Groups la and lb had also a relatively high affinity for the virus.
  • a slightly weaker binding affinity had the group la sequence HWSFGIF (SEQ ID NO: 1 1 :) and the lc sequence PWYFFGA (SEQ ID NO:54:).
  • the clones that were selected with the MAb 1 C9 could be competed with the MAb 1 C9 itself totally and the MAbs 4G2 and 5A2 had a slight inhibitory effect on the binding of the group lib clones whether selected with 1 C9 or 4G2. Finally, because the phage clones did not show any significant binding to MAbs that were used for their elution their specificities were shown to be restricted to Puumala virus glycoproteins.
  • the phage clone CHWMFSPWC (SEQ ID NO:2:) itself inhibited Puumala virus infection completely with 1 x 1 ⁇ " O TUs (about 1 nM) and the inhibition leveled off at about 1 0** TUs (Table V), which is about 1 nM and the concentration of phage that saturates the binding sites of the virus on a microliter well coated with 1 5-25 ng of virus. Maximum inhibition (approximately 60 %) was achieved with 2.5 ⁇ M peptide solution of a synthetically produced cyclic peptide CHWMFSPWC-NH 2 (SEQ ID N0:2:) as shown in Figure 4.
  • the negative control was the CX7C library, which as all of the clones, stimulated Puumala infection at amounts of 10 ⁇ TU amounts.
  • the observed stimulation was the same or higher with the selected clones and highest with the group lb and lc inhibitory sequences.
  • one of ' the peptides CSWIFWEYC (SEQ ID NO:20:) in group Id showed a stimulation that increased with the phage concentration up to 2-fold ( 10 ⁇ TU).
  • a high level of stimulation was observed with the most inhibitory clone CHWMFSPWC (SEQ ID NO:2) with 10 TUs.
  • Consensus sequences and the functionally conserved residues of the selected clones Alignment of the sequences allowed two main consensus sequences as depicted in Table V.
  • the consensus I had the sequence XWXFXXX (SEQ ID NO :39:) where particularly the residues 5-7 are highly variable.
  • the consensus II had the sequence XXXWPXX (SEQ ID NO:42:). On the basis of biochemical data the consensus II could be divided into two subgroups.
  • Table Vila summarizes cross-reactivities of the 5A2, 1 C9 and 4G2 MAbs, which have been done in ELISA against G 1 and G2 proteins separately.
  • the MAb 5A2 of the G 1 -a class does not cross-react with the other MAbs.
  • MAb 1 C9 of the G2-a class blocks the binding of MAb 4G2 of the G2-a2 class completely, whereas the MAb 4G2 that otherwise appears to have the broadest specificity does not block the binding of the 1 C9 MAb just as well.
  • the MAb 4G2 partly precipitates G1 .
  • the outcome of the peptide selection was fitted into the MAb classes (Table Vllb) .
  • the 4G2 selected consensus I consensus lla would fall into the G2-a class.
  • the consensus Mb would instead belong primarily to the G2-a2 class but it would also be shared with the G2-a.
  • the 5A2 antibody was blocking the 4G2 bindings of consensus I and lla and behaved with the lib almost identically with the 4G2. This could be due to a steric hindrance of a closely located 5A2 binding site rather than a shared epitope.
  • Respiratory syncytial virus a major virus pathogen as target pathogen
  • RSV respiratory syncytial virus
  • RNA genome RNA genome and is an enveloped virus (Piedra et al., 1 997).
  • RSV belongs to the genus Pneumovirus of Paramyxoviridae family which are monosegmented RNA viruses, they have two transmembrane envelope proteins, the glycoproteins F, which is fusion active and G, which attaches the virus to cell.
  • Paramyxoviridae viruses bud across plasma membranes and thus carry host cell membrane bilayers, which align the virus matrix protein (M) .
  • M virus matrix protein
  • the remaining gene products are the nucleoprotein (N), phosphoprotein (P), large polymerase complex (L), a strongly hydrophobic small protein (SH), a small envelope protein (M2) and two nonstructural proteins (NS1 and NS2).
  • N nucleoprotein
  • P phosphoprotein
  • L large polymerase complex
  • SH strongly hydrophobic small protein
  • M2 small envelope protein
  • NS1 and NS2 two nonstructural proteins
  • N nucleoprotein
  • P phosphoprotein
  • L large polymerase complex
  • SH strongly hydrophobic small protein
  • M2 small envelope protein
  • NS1 and NS2 two nonstructural proteins
  • the RSV neutralizing epitopes are mainly found on the F and the G glycoproteins of which the F protein is more important in induction of cross-pro ⁇ tective immunities and possibly also a better and safer target than G protein in development of protective therapies (Andersson et al., 1 985; Walsh et al., 1 987; Taylor et al, 1 992; West et., al., 1 994; Hancock et al., 1 996) .
  • F1 and F2 disulfide-linked subunits
  • F1 and F2 disulfide-linked subunits
  • the fusions are initiated by interaction of a hydrophobic domain of F protein with cell membrane.
  • Antibodies that bind to F protein are highly effective also in prevention of infection, as well as in antibody dependent clearance of an established infection. Studies propose that the most efficient protection and fusion-inhibition activity of MAbs are linked. Thus, binding of antibodies to the known fusion domains would provide reliable protections.
  • RSV provides an excellent target for our selection method, which aims to obtain peptides or molecules that reliably mimick functions of particular MAbs.
  • neutralizing MAbs that bind F protein phage-displayed peptides, which had been attached on surface of alive RSV could effectively be enriched.
  • Two different phage displayed peptide libraries were used in this study but only one gave the desired result.
  • RSV virus preparation and the virus neutralizing antibodies were kindly provided by Matti Waris (Department of Virology, University of Turku, Finland) .
  • the inoculum RS virus was prepared in Hep 2 cells and had the titer of 1 ⁇ 6 plaque forming units (PFU) per ml.
  • the partially purified RSV was ob- tained by pelleting the clarified cell culture supernatants through sucrose cushions, the titer of the virus was 10 8 PFU/ml and its protein concentration 1 ,9 mg/ml.
  • Amplified libraries with titers of 1 0 ⁇ 2 transforming units (TU) of phage/ml were diluted five times in the blocking buffer and 1 00 ⁇ l of the sample taken per well, the plates were incubated for 1 h at + 37 °C.
  • the wells were first washed 5 times with 0.5 % Tween 20 in PBS, 3 times briefly and then 2 times by vigorously shaking the plates for 5 minutes. The following 5 washes were made with the blocking buffer at + 37 °C, first 3 washes for 5 min and the last 3 for 1 5 min.
  • the phages were eluted with 10 ⁇ g antibody/100 ⁇ l blocking buffer-for 1 h at + 37 °C and the titer of eluted phages was determined, the 100 ⁇ l eluent was amplified for further experiments.
  • MAb elution was followed by a 100 ⁇ l HCl elution (0.1 M HCI-glycine pH 2.2, 1 % BSA) and results of elutions compared, As a control, elution with blocking buffer was performed.
  • For the second panning all phages that were eluted from RSV containing wells with MAb 1 30-8F, MAb 101 and HCl respectively were combined. The second panning was performed only against the inoculum virus (results not presented).
  • Binding assays were done essentially as in Puumala virus panning, Microtiter wells were coated with 1 90 ng of inoculum RSV or 80 ng of F protein in 100 ⁇ l of PBS, blocked with 1 % BSA and 0.05 % Tween 20 in PBS and washed with 0.05 % Tween 20 in PBS (PBST) . Antibodies and phages were diluted in, the blocking buffer and conjugates in PBST.
  • the F-protein of RSV-A (Randall) strain had been immunoaffinity purified with an other MAb than the previously named and was given as 80 ⁇ g/ml 50 mM Tris pH7.8, 0.02 % Sodium Azide. The amount of F-protein normally used in EIA by the supplier was 6 ng/well. However, the epitopes of MAbs 1 30-8F and 101 had been lost in these protein preparations and could not be detected at 80 ng/ml amounts.
  • Table I The human and bank vole monoclonal antibodies used in this study; specificities, neutralizing properties and anti-idiotypic antibody activities.
  • Table VA Comparison of the Puumala virus binding and inhibiting sequences eluated from the CX7C library with neutralizing antibodies 4G2 and IC9.
  • the empty spaces refer to a similarity with the residue above. Parenthesis describes a discontinuity in the alignment.
  • Table VB Comparison of the Puumala virus binding and inhibiting sequences eluated from the CX7C library with neutralizing antibodies 4G2 and 1 C9.
  • the empty spaces refer to a similarity with the residue above.
  • the regions specific for 4G2 and 1 C9 binding are underlined and the consensus sequence shared between them indicated in the overall consensus.
  • Table VI Summary of effector and binding features of conserved and variable residues of the different clones by initial studies.
  • the underlined sequences define inhibition (XXXXXPW), high binding affinity (XWXFXXX and LFXXXX) .
  • Table VIII Biopanning of partially purified (pRSV) versus inoculum RSV (iRSP) . Phage titers of input and eluted CX7C Or X2CX12CX2 library phages in transforming units.
  • Recombinant human respiratory syncytial virus (RSV) monoclonal antibody Fab is effective therapeutically when introduced directly into the lungs of RSV-infected mice.
  • RSV respiratory syncytial virus
  • a peptide isolated from phage display libraries is a structural and functional mimic of an RGD-binding site on integrins. J. Cell BioL 1 30, 1 189-1 196.
  • Temonen M., Vapalahti, O., Holth ⁇ fer, H., Brummer-Korvenkontio, M., Vaheri, A., and Lankinen, H., (1 993) Susceptibility of human cells to Puumala virus infection. J. Gen. Virol. 74, 51 5-51 8.
  • MOLECULE TYPE peptide (XI) SEQUENCE DESCRIPTION SEQ ID NO:7.

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Abstract

La présente invention concerne un procédé pour sélectionner des substances inhibitrices pathogènes présentant une forte affinité et un effet neutralisant par élution compétitive à l'aide de substances neutralisantes. Ce procédé et ce système permettent d'effectuer des études comparatives des médicaments pour fournir des substances prophylactiques et/ou protectrices, et actives sur le plan thérapeutique. Ils permettent également d'élaborer des thérapies de combinaison ainsi que des diagnostics de pathogènes. L'invention concerne aussi des procédés pour identifier les caractéristiques mimotypes du site de neutralisation des pathogènes, particulièrement des virus enveloppés, tels que l'hantavirus, le virus respiratoire syncytial, etc. L'invention concerne aussi des ligands pouvant être obtenus par le procédé ainsi que des séquences consensus et des parties et des répétitions de ces ligands prévues pour être utilisées dans les trousses d'analyse contenant des bibliothèques de ligands de combinaison comprenant les ligands sélectionnés selon ce procédé.
PCT/FI1997/000339 1996-05-30 1997-05-30 Procede pour selectionner des substances inhibitrices pathogenes cibles et trousses d'analyses prevues pour etre utilisees avec ces substances WO1997045743A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU29650/97A AU2965097A (en) 1996-05-30 1997-05-30 A method for selecting target pathogen inhibiting substances and test kits for use therein

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FI962269 1996-05-30
FI962269A FI962269A7 (fi) 1996-05-30 1996-05-30 Järjestelmä taudin aiheuttajia estävien peptidien seulomiseksi ja taudin aiheuttajien neutralisointikohtien kuvaamiseksi

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WO1997045743A1 true WO1997045743A1 (fr) 1997-12-04

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PCT/FI1997/000339 WO1997045743A1 (fr) 1996-05-30 1997-05-30 Procede pour selectionner des substances inhibitrices pathogenes cibles et trousses d'analyses prevues pour etre utilisees avec ces substances

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AU (1) AU2965097A (fr)
FI (1) FI962269A7 (fr)
WO (1) WO1997045743A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000014216A1 (fr) * 1998-09-04 2000-03-16 Zk Pharmaceuticals, Inc. Technique de selection des peptides inhibant la liaison d'une proteine virale de surface a un recepteur de surface cellulaire
WO2001040265A3 (fr) * 1999-12-02 2002-07-04 Vi Technologies Inc Procede d'identification d'un ligand destine a une molecule cible
WO2005118621A3 (fr) * 2004-04-16 2006-08-10 Genentech Inc Modulateurs omi pdz

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996004557A2 (fr) * 1994-08-03 1996-02-15 Dgi Technologies, Inc. Triages specifiques de cibles et leur utilisation permettant la decouverte de petits groupements pharmacophores moleculaires organiques

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996004557A2 (fr) * 1994-08-03 1996-02-15 Dgi Technologies, Inc. Triages specifiques de cibles et leur utilisation permettant la decouverte de petits groupements pharmacophores moleculaires organiques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEM. REV., Volume 97, 1997, GEORGE P. SMITH et al., "Phage Display", pages 391-410. *
DIALOG INFORMATION SERVICES, File 34, SciSearch, Dialog Accession No. 15592784, HEISKANEN T. et al., "Phage-Displayed Peptide Targeting on the Puumala Hantavirus Neutralization Site"; & JOURNAL OF VIROLOGY, May 1997, Vol. 71, No. 5, p3879-3885. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000014216A1 (fr) * 1998-09-04 2000-03-16 Zk Pharmaceuticals, Inc. Technique de selection des peptides inhibant la liaison d'une proteine virale de surface a un recepteur de surface cellulaire
WO2001040265A3 (fr) * 1999-12-02 2002-07-04 Vi Technologies Inc Procede d'identification d'un ligand destine a une molecule cible
WO2005118621A3 (fr) * 2004-04-16 2006-08-10 Genentech Inc Modulateurs omi pdz

Also Published As

Publication number Publication date
FI962269L (fi) 1997-12-01
FI962269A7 (fi) 1997-12-01
FI962269A0 (fi) 1996-05-30
AU2965097A (en) 1998-01-05

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